Herbert Steinbeisser:A life with the Xenopus embryo

Herbert Steinbeisser was a developmental biologist completely immersed in science. Of a cheerful disposition and constant good humor, he was the best collaborator one could hope for. When such a nice, kind colleague is taken by cancer at age 56 it seems so unjust. Yet, his life is an illustration of how wonderful a life in science can be and we would like to relate it here.

Ketsch, Heidelberg and EcuadorHerbert was born in Ketsch, a small village near the Rhine river where he grew up fishing for pike with his grandfather. He did his Ph.D. at the nearby University of Heidelberg with Michael Trendelenburg studying the transcription of protein coding genes in Xenopus oocytes (Steinbeisser et al., 1989). After graduating he won an Alexander von Humboldt Foundation postdoctoral fellowship to work with Eugenia del Pino at the Pontificia Universidad Católica in Quito, Ecuador. There, in 1990, he worked with the giant oocytes of the marsupial frog Gastrotheca, which carries fertilized eggs on its back (del Pino et al., 1986). Herbert loved Ecuador and maintained a long-standing exchange of students.

CaliforniaOne day one of us (EDR) received a letter from Eugenia del Pino saying she had this German postdoc that wanted to set up Richard Harland’s new whole-mount in situ hybridization method and could he come to Los Angeles to use our Xenopus embryos. He eventually stayed for four highly productive years. At that time Ken Cho and Bruce Blumberg had just cloned a homeobox gene called goosecoid. In one of his first in situ experiments Steinbeisser observed a region of staining on the dorsal side of a Xenopus gastrula. We all congregated over the dissection microscope as the color developed. It was a great moment in embryology, for it was the first time that Spemann’s organizer had been directly visualized (Cho et al., 1991). Previously, the existence of Spemann’s organizer was deduced from the inductive activity of transplanted dorsal blastopore lips, but now we had a gene that marked this tissue. One of Herbert’s favorite experiments was to dorsalize an embryo

IN MEMORIAM

Int. J. Dev. Biol. 58: 299-302 (2014)doi: 10.1387/ijdb.140117ed

by lithium chloride (LiCl) treatment (which mimicks the early canonical Wnt signal by inhibiting GSK3, leading to an embryo in which the entire mesoderm becomes organizer) or to ventralize it by irradiation with ultraviolet (UV) light; goosecoid behaved exactly as expected for a Spemann organizer gene (Cho et al., 1991). With an easy-going and optimistic personality Steinbeisser was always ready to collaborate even in the most technically difficult experiments. Martin Blum, who became a life-long fast friend of Herbert’s, had cloned the mouse homologue of goosecoid and we had found that it was expressed at the anterior primitive streak in 6.75 day mouse embryos. I presented this at a meeting and it caused great consternation, for the Spemann organizer of the mouse was presumed to reside on the node, a structure that appears later. Fortunately, Azim Surani suggested that we could test the inductive potential of this region by transplantation into Xenopus embryos. Mice breed at night, and each female has embryos at a wide range of stages. We embarked on a huge experiment in which Herbert prepared Xenopus gastrula stage embryos, Martin Blum dissected pregnant mice to extract embryos at the exact stage, and EDR transplanted the anterior tip of the primitive streak or its posterior region into the blastula cavity of Xenopus embryos (by the Einsteck method). We were at it all day long, and by the time we had gone through all the mice it was midnight, and we went to enjoy a beer in camaraderie at a popular restaurant and bar near UCLA. It was one of those magical days that make scientific research so worthwhile. Two days later we were rewarded with some beautiful inductions of head structures, of Xenopus origin, induced specifically by the goosecoid-expressing region of the mouse gastrula (Blum et al., 1992). Steinbeisser’s main project was to study the ventralizing effect of Bone Morphogenetic Protein 4 (BMP4), which had the opposing effect to that of goosecoid mRNA (Steinbeisser et al., 1995). In loss-of-function studies he showed that antisense RNA for BMP4 could

Fig. 1. Herbert Steinbeisser (1958-2014). During a cruise on the river Mosel, on occasion of the 12th International Xenopus Conference in Leiwen, Germany. Photo by Christof Niehrs, (September 2008).

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reverse the ventralizing effects of UV irradiation, and that gsc antisense could inhibit the dorsalizing effects of LiCl treatment. These two antisense RNAs worked very well, as had been seen before by Herbert Jäckle with Drosophila Krüppel. Unfortunately, studies in zebrafish claimed that other antisense RNAs had non-specific effects, and it became very difficult to publish any studies using anti-sense RNA loss-of-function. Thus, very valuable reagents were lost to Xenopus researchers due to scientific fashion. The observed results (Steinbeisser et al., 1995) were probably the result of the formation of interfering RNAs, but RNAi had not been discovered at the time. Our studies on BMP4 were the result of a very fruitful collaboration between Herbert and a sabbatical visitor from Jerusalem, Abraham Fainsod, with whom he kept a long lasting friendship (Fainsod et al., 1994). Because Steinbeisser was working on BMP4, he was in a good position to make rapid progress when the BMP antagonist chordin was cloned as a downstream target of goosecoid in the lab (Sasai et al., 1994 and 1995). One anecdote worth telling is how Herbert received the Mayor of Ketsch in Los Angeles. In California we sometimes have the most wonderful sunsets. One such gorgeous day the two authors looked at each other and decided they just had to watch this sunset from a bar at the Santa Monica Pier, about 5 miles from UCLA. We sat right by the water, and overheard German spoken behind us. There were Herbert Steinbeisser, the Mayor of Ketsch and a fellow citizen. We bought them a round, and they were so happy with the glittering on the Pacific Ocean and grateful for conversation in German that they invited us to fish for pike anytime at Ketsch village. Obtaining a permit to do this if you are not a local is probably next to impossible; this was a great honor although the offer was never taken up. Herbert was completely dedicated to science yet also a very social person who knew how to enjoy the simple pleasures of life. The most important event of this postdoctoral stay was meeting Sarah Cramton, a Ph.D. student in the lab right across the corridor at UCLA. They married upon his return to Germany, had two children, Karl and Marta. This was a happy marriage.

TübingenIn 1995 Herbert became a Group Leader at the Max Planck Institute for Developmental Biology in the Department of Peter Hausen, a phage geneticist turned a strong leader of Xenopus research. His first paper there was a collaboration with Stephan Schneider, a graduate student of Hausen. They made the important discovery that endogenous b-catenin protein translocated inside nuclei in the dorsal side of the midblastula embryo, both in Xenopus and zebrafish (Schneider et al., 1996). In this study we can see Steinbeisser’s imprimatur for he used LiCl to show stabilization of b-catenin all around the embryo and UV irradiation to block nuclear translocation on the dorsal marginal cells. This paper was a landmark study in embryology. The dorsal localization of b-catenin is caused by a cortical rotation of the egg cytoplasm, a mechanism to which Herbert was to return (Medina et al., 1997; Ding et al., 1998). Steinbeisser’s interest turned to Frizzled seven-transmembrane proteins, which had been recently discovered by Roel Nusse and Jeremy Nathans as the long sought Wnt receptors. In a series of papers he explored the role and structure-functional aspects of Fz7 during Xenopus development (Swain et al., 2001; Medina and Steinbeisser, 2000; Medina et al., 2000). This culminated in the discovery, together with Rudi Winklbauer, that Fz7/PKC signaling regulates sorting out of early involuted anterior endomesoderm from ectoderm. This comes about by a switch in cell adhesion and is essential in Xenopus gastrulation (Winklbauer et. al., 2001). The paper featured an elegant explant assay, where embryonic cells are placed on naïve ectoderm and either sink-in or stay put, the type of experiment that makes Xenopus such a unique system. This study set the stage for a series of follow-up papers on tissue separation, notably involving the then poorly understood protocadherins. These transmembrane proteins play an important role in cell adhesion and morphogenesis. In Tübingen, and later in Heidelberg, Herbert showed that paraxial protocadherin (PAPC) regulates tissue separation and that it is a signaling molecule, which activates Wnt/Planar Cell Polarity (PCP) signaling (Medina et al., 2004; Wang et al., 2008; Kraft et al., 2012) and inhibits canonical Wnt signaling by sequestering casein kinase 2 beta (Kietzmann et al., 2012). Steinbeisser’s studies on tissue separation renewed interest in an extracellular matrix region of the Xenopus embryo that separates the endomesoderm from the ectoderm called Brachet’s cleft (Gorny and Steinbeisser, 2012). This narrow region is now thought to provide a signaling highway during patterning of the gastrula.

Fig. 2. Herbert Steinbeisser and wife Sarah Cramton, with Ana and Eddy De Robertis in Pacific Palisades, California. Photo by Marta Steinbeisser (August 2013).

Heidelberg and return to Ketsch In 2002 Herbert was appointed professor by his alma mater, Heidelberg University, where he joined the Institute of Human Genetics. He was able to return to his roots, and entirely renovated the home of his grandparents in the tiny village of Ketsch. It was a beautiful house full not only with his own two children but also with kids from the neighbors that would wander in and out, as did their cats. This was a very happy home. The human geneticists in Heidelberg realized that they needed a tractable animal model system to study the mechanisms of inborn human malformations and syndromes. In Steinbeisser they hit on the ideal colleague because a distinguishing feature was his strong collaborative nature, which manifested itself already in his postdoc days. Modest and optimistic by nature, he was always ready to contribute to studies with his Xenopus or Wnt expertise, even if it meant a less visible author position for himself. For example he coauthored a series of papers with Gudrun Rappold on the Leri-Weill and Turner syndrome homeobox gene SHOX (Hoffman et al., 2013; Puskaric et al., 2010; Blaschke et al., 2003), and with Jochen Zschocke on the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (Rauschenberger et al., 2010; Deutschmann et al., 2014), congenital defects of which lead to childhood neurodegeneration. Between 2008-2014 Steinbeisser was the leader of a Wnt research network (Wnt-FOR) funded by the German Research Foundation (DFG). The Wnt-FOR was a success story that is inextricably linked to Herbert's name, even if he was far too modest to ever articulate this. The Wnt-FOR brought together scientists from the Heidelberg, Karlsruhe and Mannheim area, who study different aspects of Wnt biology. The Heidelberg area is fortunate to have a critical mass of colleagues working in this field (including A. Aulehla, M. Boutros, G. Davidson, D. Gradl, S. Hardt, T. Holstein, A. Martin-Villalba, C. Niehrs, S. Özbek, S. Scholpp, H. Steinbeisser and D. Wedlich). Herbert not only made major contributions to Wnt signaling in early Xenopus development, but above all he had a special touch to integrate a heterogeneous group of scientists and balance varying interests. He had an open ear for everybody’s concerns, particular needs, and interests, and everyone felt respected and uplifted by him. Steinbeisser successfully steered the research group through the cliffs of the installation grant, coordinated its successful extension, and organized two very successful Wnt symposia in Heidelberg that gave international visibility to the Wnt-FOR. The Wnt-FOR trained more than a dozen young scientists, created a series of Wnt studies in development and beyond, and established the Rhine-Neckar area as an internationally visible Wnt research hub. The last few months, already in poor health, Herbert tirelessly worked to transform and expand the Wnt-FOR beyond the boundaries of Heidelberg to a DFG-Transregio initiative. Colleagues who witnessed his deteriorating health admired how stoically and persevering he pursued this goal, undeterred by his fate. Fortunately, he was able to complete and submit the concept paper to the funding agency before passing away.The Heidelberg Wnt community will long benefit from his relentless commitment. A donation fund was established in Herbert Steinbeisser’s name, which will support young scientists of the Pontificia Universidad Católica del Ecuador, where Herbert spent happy months doing research on Gastrotheca. Herbert will be missed by many scientific friends that collaborated with him throughout the years, but his scientific contributions and personal legacy will remain.

Eddy M. De Robertis and Christof NiehrsLos Angeles, USA and Mainz, Germany

References

BLASCHKE RJ, TÖPFER C, MARCHINI A, STEINBEISSER H, JANSSEN JW, RAPPOLD GA (2003). Transcriptional and translational regulation of the Leri-Weill and Turner syndrome homoebox gene SHOX. J. Biol. Chem. 28: 47820-47826.

BLUM M, GAUNT SJ, CHO KWY, STEINBEISSER S, BLUMBERG B, BITTNER D, DE ROBERTIS EM (1992). Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69: 1097-1106.

CHO KWY, BLUMBERG B, STEINBEISSER H, DE ROBERTIS EM (1991). Molecular Nature of Spemann's Organizer: the Role of the Xenopus Homeobox Gene goosecoid. Cell 67, 1111-1120.

DEL PINO EM, STEINBEISSER H, HOFMANN A, DREYER C, CAMPUS M, TRENDELENBURG MF (1986). Oogenesis in the egg-brooding frog Gastrotheca riobambae produces large oocytes with fewer nucleoli and low RNA content in comparison to Xenopus laevis. Differentiation 32: 24-33.

DEUTSCHMANN AJ, AMBERGER A, ZAVADIL C, STEINBEISSER H, MAYR JA, FEICHTINGER RG, OERUM S, YUE VW, ZSCHOCKE J (2014). Mutation of knock-down of 17-hydroxysteroid dehydrogenase type 10 cause loss of MRPP1 and impaired processing of mitochondrial heavy strand transcripts. Hum. Mol. Genet. 23: 3618-3628

DING X, HAUSEN P, STEINBEISSER H (1998). Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction. Mech. Dev. 70: 15-24.

FAINSOD A, STEINBEISSER H, DE ROBERTIS EM (1994). On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13: 5015-5025.

GORNY AK, STEINBEISSER H. (2012), Brachet’s cleft: a model for the analysis of tissue separation in Xenopus. Wiley Interdiscip. Rev. Dev. Biol. 1: 294-300.HOFFMANN S, BERGER IM, GLASER A., BACON C, LI L, GRETZ N, STEINBEISSER H, ROTTBAUER W, JUST S, RAPPOLD G. (2013). Islet1 is a

direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia. Basic Res. Cardiol. 108: 339.KIETZMANN A, WANG Y, WEBER D, STEINBEISSER H (2012). Xenopus paraxial protocadherin inhibits Wnt/b-catenin signaling via casein 2. EMBO

Rep. 13: 129-134.KRAFT B, BERGER CD, WALLKAMM V, STIENBEISSER H, WEDLICH D (2012), Wnt-11 and Fz7 reduce cell adhesion in convergent extension by

sequestration of PAPC and C-cadherin. J. Cell Biol. 198: 695-709.MEDINA A, STEINBEISSER H (2000). Interaction of Frizzled 7 and Dishevelled in Xenopus. Dev. Dyn. 218: 671-680.MEDINA A, WENDLER SR, STEINBEISSER H (1997). Cortical rotation is required for the correct spatial expression of nr3, sia and gsc in Xenopus em-

bryos. Int. J. Dev. Biol. 41: 741-745.

MEDINA A, REINTSCH W, STEINBEISSER H (2000). Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on

early patterning and morphogenesis. Mech. Dev. 92: 227-237.MEDINA A, SWAIN RK, KUERNER KM, STEINBEISSER H (2004). Xenopus paraxial protocadherin has signaling functions and is involved in tissue

separation. EMBO J. 23: 3249-3258.PUSCARIC S, SCHMITTECKERT S, MORI, AD, GLASER A, SCHNEIDER KU, BRUNEAU BG, BLASCHKE RJ, STEINBEISSER H, RAPPOLD G. (2010).

Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart. Hum. Mol. Genet. 19: 4625-4633. RAUSCHENBERGER K, SCHÖLER K, SASS JO, SAUER S, DJURIC Z, RUMIG C, WOLF NI, OKUN JG, KÖLKER S, SCHWARZ H, FISCHER C,

GRZIWA B, RUNZ H, NÜMANN A, SHAFQAT N, KAVANAGH KL, HÄMMRLING G, WANDERS RJ, SHIELD JP, WENDEL U, STERN D, NAWROTH P, HOFFMAN GF, BARTRAM CR, ARNOLD B, BIERHAUS A, OPPERMAN U, STEINBEISSER H, ZSCHOCKE J (2010). A non-enzymatic function of 17-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival. EMBO Mol. Med. 2: 51-62.

SASAI Y, LU B, STEINBEISSER H, GEISSERT D, GONT LK, DE ROBERTIS EM (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79: 779-790.

SASAI Y, LU B, STEINBEISSER H, DE ROBERTIS EM (1995). Regulation of neural induction by the Chd and BMP-4 antagonistic patterning signals in Xenopus. Nature 376, 333-336.

SCHNEIDER S, STEINBEISSER H, WARGA RM, HAUSEN P (1996). Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech. Dev. 57: 191-198.

STEINBEISSER H, ALONSO A, EPPERLEIN HH, TRENDELENBURG, MF (1989). Expression of mouse histone H10 promoter sequences following microinjection into Xenopus oocytes and embryos. Int. J. Dev. Biol. 33: 297-304.

STEINBEISSER H, FAINSOD A, NIEHRS C, SASAI Y, DE ROBERTIS EM (1995). The role of gsc and BMP-4 in dorsal-ventral patterning of the marginal zone in Xenopus: a loss-of-function study using antisense RNA. EMBO J. 14: 5230-5243.

SWAIN RK, MEDINA A, STEINBEISSER H (2001). Functional analysis of the Xenopus frizzled 7 protein domains using chimeric receptors. Int. J. Dev. Biol. 45: 259-264.

WANG Y, JANICKI P, KÖSTER I, BERGER CD, WENZL C, GROSSHANS J, STEINBEISSER H (2008). Xenopus Protocadherin regulates morphogenesis by antagonizing Sprouty. Genes Dev. 22: 878-883.

WINKLBAUER R, MEDINA A, SWAIN RK, STEINBEISSER H (2001). Frizzled-7 signalling controls tissue separation during Xenopus gastrulation. Nature 413: 856-860.

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